Method for producing interleukin 1 receptor antagonists, a therapeutic activity protein, from body fluids

ABSTRACT

The present invention relates to a method for producing a prophylactically or therapeutically active protein, the syringe being filled with a body fluid and incubated, and the prophylactically or therapeutically active protein subsequently being formed in the body fluid.

[0001] The present invention relates to a method for producingprophylactically or therapeutically active proteins and to the meansemployed therefor, in particular syringes.

[0002] Therapeutically active proteins such as erythropoietin, insulinor interferons have been known for a long time. Many of these proteinshave already been approved as medicaments and are accordingly employedfrequently. Because of the large costs associated with the developmentand approval of these medicaments, however, there is a need for simpleand cost-effective alternatives for the preparation of therapeuticallyactive proteins. In addition, not all therapeutically active proteinsare approved as medicaments. Nevertheless, however, there is frequentlya need for these proteins also to be administered to the patient.Particularly important in this connection, because they are presumed tobe well tolerated by the body, are autologous proteins, that is to saythose intrinsic to the body. These proteins include the interleukin 1receptor antagonist, interleukin 4, interleukin 10 and the type I ortype II tumor necrosis factor receptor. Proteins intrinsic to the bodymoreover have the advantage that the natural post-translationalmodifications such as glycosylations are already present. This is notthe case with most normally obtainable recombinant proteins because theyare produced in prokaryotic hosts.

[0003] Stimulation of monoctyes by adherent immunoglobulin G to form theinterleukin 1 receptor antagonist is described by Arend and Leung inImmunological Reviews (1994) 139, 71-78 and Moore et al. in Am. J.Respir. Cell Mol. Biol. (1992) 6, 569-575. Andersen et al. inAutommunity (1995) 22, 127-133 explains that the therapeutic effect ofimmunoglobulin G to be observed in vivo cannot be attributed to anenhanced formation of interleukin 1 receptor antagonist, and that the invitro formation of the interleukin 1 receptor antagonist (IRAP, IL-1Ra)by monocytes depends on serum and plasma constituents adsorbed onpolypropylene. The therapeutic use of adsorbed serum and plasmaconstituents to stimulate the formation of therapeutically interestingproteins in therapies is not only very costly but also involves the riskof contamination with infectious particles with which the serum andplasma constituents may be contaminated. Methods for producing IL-1Rawhich can be employed directly in the therapy without using adsorbedserum and plasma constituents are not described in the aforementionedpublications.

[0004] The technical problem on which the present invention is based isthus to provide methods and means for producing IL-1Ra which serve assafe, cost-effective alternatives which can be carried out quickly forthe use and for the production of conventional pharmaceutical products.

[0005] The invention solves this problem by providing a method forproducing IL-1Ra in a syringe made of glass, quartz or a plastic, thesyringe being filled with a body fluid from an organism, for example ahuman or animal body, and incubated, and the IL-1Ra being formed.

[0006] A preferred embodiment of the present invention provides for theinternal structure of the syringe to consist of a special material, inparticular a glass, plastic, quartz and/or corundum, whose surface is,in a particularly preferred embodiment of the invention, modified, inparticular with the aid of a corrosive agent, for example an acid or analkali, in particular chromic/sulfuric acid, and subsequently, afterremoval of the agent and washing the syringe where appropriate, that isto say in a particularly preferred manner, the surface of the internalstructure of the syringe is sterilized, in particular by autoclaving.The syringe is then filled with a patient's body fluid and incubated,and IL-1Ra is formed thereby. The body fluid enriched with the proteincan then be reinjected into the patient, for example into a diseasedjoint. However, to increase the purity of the IL-1Ra which has beenformed, the body fluid is preferably centrifuged and the supernatant issterilized by filtration, divided into aliquots and stored for a latertreatment. The invention thus provides in a preferred embodiment in afirst step of the method for the surface of the internal structure ofthe syringe to be modified, in particular with the aid of a corrosiveagent, such as an acid or an alkali, in particular chromic/sulfuricacid, and then, if desired, the surface of the internal structures ofthe syringe is sterilized, in particular by autoclaving. A drying can beprovided before and/or after the sterilization. After the modificationand the sterilization which takes place where appropriate, the syringeis filled in a second step of the method with a body fluid, inparticular blood, lymph, saliva or urine, and incubated. The body fluidis preferably taken from the patient directly with the syringe. The,preferably modified, surface of the internal structure of the syringeinduces in the body fluid specifically, to an extent which differsquantitatively depending on the material employed, the internalstructure of the syringe, modification employed, in particular etchingof the internal structure, sterilization employed, in particularautoclaving, and body fluid employed, the formation of IL-1Ra which,accordingly, becomes enriched or is formed in the body fluid present inthe syringe. The body fluid enriched in this way can be stored sterilein the syringe and be returned to the patient as required directlywithout further treatment or, preferably, after centrifugation and/orsterilization by filtration.

[0007] The invention thus also relates to a method for the prophylacticor therapeutic treatment of the human or animal body, for example forthe treatment of rheumatism, osteoarthritis and/or back symptoms,wherein a body fluid, for example blood, is taken from the human oranimal body by means of a syringe according to the present invention,this body fluid is incubated in the syringe, and IL-1Ra is formed orenriched thereby, and the body fluid is returned using this syringe tothe same or a different human or animal body.

[0008] The IL-1Ra which is formed can advantageously be modified, forexample glycosylated. It is self-evident that the invention alsoencompasses the formation of other modifications or variants of IL-1Ra,such as truncated forms, mutants or other derivatives.

[0009] In conjunction with the present invention, an internal structureof a syringe means any region or any structure of the syringe which ispresent in its interior, that is to say in the sample reception regionand may come into contact with the body fluid to be received. Theinternal structure of a syringe is particularly advantageously its innersurface, preferably a surface with a texturing to increase the surfacearea. It is self-evident that the present invention can also be carriedout using a commercially available syringe without a specialconfiguration in its internal cavity. In such a case the internalstructure is the inner surface of the syringe barrel and the part of thepiston present in the barrel. The internal structure can, in aparticularly preferred embodiment, additionally be formed by articlesintroduced into the interior of the syringe, such as particles, spheres,beads, gels, glass wool, corundum, quartz, sand, plastic or glassgranules or powder or the like in order to enlarge the internal surfacearea of the syringe and thus provide a larger inducing surface. Thematerial of which the internal structure consists or which is present inthe internal structure can be a material different from that of whichthe remainder of the syringe consists. For example, the syringe mayconsist of plastic and parts of its internal structure may, for example,consist of glass granules.

[0010] Additional structures of these types, such as, for example, glassbeads with a diameter of from 1 to 5 mm, should, according to apreferred embodiment of the invention, however occupy not more than 50%of the internal volume of the syringes employed. The syringes employedmay be, for example, 10 to 100 ml syringes.

[0011] In connection with the present invention, a modified surface ofan internal structure of the syringe means a surface which has beentreated with a corrosive agent and which is capable of inducing and/orenhancing the formation of IL-1Ra in a body fluid of a human or animal.The modified surface may be distinguished by a particularly greatcleanliness, that is to say substantial or complete absence ofcontaminants and/or a chemical/physical modification of the surfaceproperties and/or structure. The modified surface of the internalstructure is preferably produced by employing an alkali or an acid, forexample chromic/sulfuric acid, in particular 20 to 80% strength,particularly preferably 50% strength, chromic/sulfuric acid. The syringeis preferably incubated with the corrosive agent, in particular thechromic/sulfuric acid, for 5 to 30 min.

[0012] After removal of the agent it is possible and preferred to carryout one or more washing steps and, where appropriate, preferably asterilization, for example by autoclaving, in particular autoclaving at100° C. to 150° C. for 20 to 60 min under a pressure of from 1 to 5 bar.After the washing and before and/or after the autoclaving it is possiblewhere appropriate also for one or more drying steps to take place at 60to 150° C., preferably 80° C., for 30 to 120 min in, for example, adrying oven.

[0013] A particularly advantageous configuration of the inventionprovides for the syringe, in particular the internal structure of thesyringe, to be produced from glass, for example quartz glass, corundum,quartz, a plastic such as polystyrene, polyethylene, polyvinyl chloride,polypropylene, or a similar material, that is to say consists of thesematerials or essentially comprises these materials or mixtures thereof.

[0014] It has surprisingly been possible to show within the framework ofthe present teaching that a syringe made of glass, and/or an internalstructure made of glass, in particular glass granules, displays aparticularly strongly inducing effect.

[0015] In a particularly preferred embodiment, heated glass is usedbefore the treatment, that is to say before removal of the body fluid,in particular glass which has been heated to 100° C.-210° C., inparticular 1700-200° C., preferably 180° C., and which is, of course,cooled before the use according to the invention. In a particularlypreferred embodiment, the glass can be in the form of glass powder orglass granules.

[0016] The internal structures may, however, also consist of quartz, forexample quartz powder or quartz sand and/or corundum, for example in theform of a suspension in water, or comprise the latter.

[0017] In a particularly preferred embodiment, these materials displayIL-1Ra-inducing properties after modification, in particular etching,has taken place where appropriate and after the sterilization has takenplace where appropriate.

[0018] A further preferred embodiment of the invention provides for theinternal structure of a syringe to be provided with a coating which canbe modified and can be sterilized, and for the modification and, whereappropriate, the sterilization to be carried out subsequently.

[0019] The present invention is advantageous inasmuch as it provides amethod which is simple to carry out and with which it is possible toproduce autologous IL-1Ra which can be produced by induction, inparticular surface induction, and can, in the form thus produced, thatis to say together with the other constituents of the fluid present inthe syringe, be readministered to the patient directly, that is to saywithout further manipulation such as, for example, transfer into anothercontainer. In a preferred embodiment, a centrifugation and/orsterilization by filtration can be provided to remove solid constituentsbefore the IL-1Ra which has been formed is administered. The use ofcommercially available, often costly, medicaments thus becomesredundant. Finally, the invention proves to be advantageous since itavoids contamination, pollution, infecticii or the like of the IL-1Rawhich derives from a medicament production taking place outside thepatient.

[0020] The present invention thus provides in a particularly preferredembodiment a method for producing the interleukin 1 receptor antagonist,where the internal structures of the syringe consist of a speciallytreated material, in particular glass, quartz or plastic, where thesurface of the internal structures of the syringe has been modified, inparticular with the aid of a corrosive agent, in particularchromic/sulfuric acid, where the surface of the internal structures ofthe syringe has, where appropriate, subsequently been sterilized, inparticular by autoclaving, and where the syringe is filled with a bodyfluid, preferably blood, and incubated, and IL-1Ra is formed andenriched in the body fluid. Inter alia because of the specialmodification of the surface of the internal structures of the syringe,in particular etching, and the special sterilization of the surface, inparticular autoclaving, the syringe of the invention is able tostimulate the monocytes present in the blood to form IL-1Ra so that thelatter becomes enriched in the blood. The blood present in the syringecan, in one embodiment of the present invention, after incubation, thatis to say after enrichment of the IL-1Ra, be returned to the patientfrom whom the blood introduced into the syringe was taken, directly,without further manipulation such as, for example, transfer.

[0021] It is possible and advantageous in a particularly preferredembodiment of the present invention to provide a centrifugation and/orsterilization by filtration of the blood present in the syringe in orderto remove solid constituents such as cells. It is then divided intoaliquots where appropriate. It is thus also provided by the inventionthat the blood can be taken from the patient by means of the syringeequipped with a specially modified and sterilized, in particularautoclaved, surface of the internal structures, the blood can beincubated in the syringe and, after formation of IL-1Ra, can be returnedto the same patient or another one with the syringe. Such a procedure isparticularly advantageous for example in the area of neuroorthopedics,that is to say, for example, for back symptoms with a neurologicalcausation. Suitable treatment for such symptoms to date has been only anintervertebral disk operation, cortisone treatment, irrigations withsaline solutions or the like. The provision by the invention of IL-1Ra,that is to say, in particular, autologous IL-1Ra, also makes treatmentof rheumatism and osteoarthritis possible in a particularly simple,cost-effective and efficacious way.

[0022] The invention provides in another embodiment for the internalstructure of the syringe additionally to be coated with anticoagulants,in particular heparin, citrate, EDTA, CPDM or CPDA. It has surprisinglybeen possible to show within the framework of the present teaching thata good induction is achieved on use of heparin as anticoagulant. It isalso possible to provide according to the invention for theanticoagulants to be employed not as coating but unbound in thecontainer, for example put in the lyophilized or liquid state into thesyringe.

[0023] However, in a particularly preferred embodiment of the presentinvention, no anticoagulant, in particular no heparin, is employed inthe syringe. This leads to a further improvement in the incubation.

[0024] The invention provides in a further preferred embodiment for theincubation of the body fluid in the syringe to be carried out over aperiod of from 12 to 72 hours, preferably 24 hours, preferably at roomtemperature, that is to say 20° C. to 41° C., in particular at 37° C.

[0025] The invention also provides in one configuration of the inventionfor the body fluid to be treated further after formation of thetherapeutically or prophylactically active protein in the body fluid, inorder, for example, to remove particular constituents of the latter, forexample blood plasma or blood platelets. This removal can in a preferredembodiment of the invention be carried out by centrifugation orfiltration.

[0026] The invention relates in a further embodiment to a method forproducing a syringe which is suitable for the in vitro induction ofprophylactically or therapeutically active proteins, in particular theinterleukin 1 receptor antagonist, where the syringe is distinguished bythe specially treated material of the internal structure of the syringe,in particular plastic or glass. The invention provides in particular forthe surface of the internal structure of the syringe to be etched by acorrosive agent, in particular an alkali or an acid, in particular usingchromic/sulfuric acid. After removal of the corrosive agent and washingof the syringe it can be provided for the surface of the internalstructure to be sterilized, in particular autoclaved. It can preferablyalso be provided for a syringe, preferably produced from glass, to beheated before use thereof, for example to 100° C. to 210° C., inparticular 170°-200° C.

[0027] The invention of course also relates to the syringe produced inthis way, which, in a particularly preferred embodiment, is producedfrom glass, quartz or plastic, in particular polystyrene, polyvinylchloride, polyethylene or polypropylene, where the syringe isdistinguished by the special treatment of the surface of the internalstructure of the syringe, in particular produced from glass, quartz orplastic, which is carried out by exposure to a corrosive agent. In apreferred manner, the syringe has been heated before use thereof,preferably to 100° C.-210° C., for example 170° C.-200° C.

[0028] The invention also relates to the use of alkali or acid, inparticular chromic/sulfuric acid, for modifying the surface of theinternal structures of the syringes of the invention, preferably made ofpolystyrene, polyvinyl chloride, polyethylene, polypropylene, quartz orglass, for the in vitro induction of prophylactically or therapeuticallyactive proteins, preferably the interleukin 1 receptor antagonist.

[0029] The invention also relates to the use of devices or substanceswhich enlarge the surface area, such as glass powder, glass granules,quartz powder, quartz sand, corundum, spheres, beads, sand etc. for usein an aforementioned method, that is to say in particular for use asinducer of IL-1Ra formation in a vessel, for example a syringe.

[0030] Further advantageous configurations of the invention are evidentfrom the dependent claims.

[0031] The invention is illustrated in detail by means of figures andexemplary embodiments.

[0032] The figures show:

[0033]FIG. 1 shows a diagrammatic representation of a syringe of theinvention.

[0034] FIGS. 2 to 12 show the formation of IL-1Ra in a syringe employedaccording to the invention under various conditions.

[0035]FIGS. 13 and 14 show the corrosive agent employed is completelyremoved by washing the syringe.

EXAMPLE 1

[0036] Production and Use of a Glass Syringe With Granules

[0037]FIG. 1 shows a 50 ml syringe 1 made of glass (Fortuna Optima,Order No. 7.102.-44, unless indicated otherwise hereinafter, thissyringe was employed in all the examples) with a piston or plunger 3,with a closure 5 which can be unscrewed and has a closure attachment 13(male Luer) and with a removable cap 7 which is disposed on the closureattachment 13 and closes the latter and has a septum. The plunger 3 hasa predetermined breaking point 15. It is thus possible to centrifuge thesyringe directly after breaking off the plunger. Glass granules 9 (glassbeads from Roth, Art. No. A 557.1) are also depicted. The size of theparticles of the granules 9 is between 1 and 3 mm in diameter, it alsobeing possible, however, to employ smaller particles, in particularlarger than 100 μm, for example glass powder. It is, of course, alsopossible to employ syringes, for example made of glass or plastic, whichhave no predetermined breaking point in the plunger.

[0038] The syringe 1 is produced by modifying the surface of theinternal structure of the syringe 1 which is new ex-factory and in itsoriginal pack and of the granules 9 which are new ex-factory and intheir original pack with the aid of a commercially availablechromic/sulfuric acid product by taking the chromic/sulfuric acidproduct into the syringe and etching the inner wall of the syringe, thatis to say inner wall of the barrel and piston, and the granules,therewith. The syringe is treated, and thus cleaned and modified, by 50%strength chromic/sulfuric acid (Merck, Darmstadt, Order No.1.02499.2500, chromic/sulfuric acid is diluted with Biochrom ultrapurewater No. L 0040 to the desired dilution) being completely drawn up andejected one to ten times, preferably three times. After the last drawingup, the syringe is sealed at the bottom and incubated in the statefilled with chromic/sulfuric acid for 5 to 30 min. The syringe piston isthen removed and thoroughly washed two to ten times, preferably fourtimes, by complete filling and draining of the syringe barrel with freshultrapure water, it being necessary to take care that filling andemptying of the washing water is complete. The syringe piston is thenimmersed in 50% strength chromic/sulfuric acid and thoroughly washedwith distilled water.

[0039] Any residues of water present in the syringe are removed bycapillary suction by swabbing the Luer connector in order to ensurerapid drying of the syringe. Pistons and syringes including any glassbeads present therein are separately sealed (Melag, Melaseal) in Melagfilm with indicator field (Melag, Melafol 1502). The syringes packed inthis way are dried in a drying oven (Melag drying sterilizer) at 80° C.for at least 60 min. The dried, packed syringes are then autoclaved at132° C. under 2 bar for 30 min (Wolf Autoclave HRM 242 II) and driedonce more at 80° C. for at least 60 min.

[0040] Before the removal of blood (see below), heparin (Liquemin N2500, heparin sodium 2500 I.U.) or citrate (ACDA) is introduced into thesyringe in order to prevent coagulation of the blood taken up later. Theuse of coagulants may prove to be advantageous in the processing ofIL-1Ra containing serum.

[0041] The syringe 1 is employed by removing blood from a patient withthe aid of an adapter which is not depicted and which connects the cap 7which can be unscrewed by means of a tubing, which is not depicted, to acannula which is not depicted. The adapter has a needle by means ofwhich the septum present in the closure attachment 13 is pierced. Theadapter is then removed and the whole blood is incubated at 37° C. for24 hours under the protection of the removable cap 7, whose septum hasclosed automatically. The incubation can take place vertically orhorizontally. If the incubation takes place vertically, the plasma isremoved through the septum and a sterile fitted filter (0.2 μm). Inaddition or alternatively, a centrifugation can be provided. If theincubation takes place horizontally, the blood is centrifuged and theplasma is removed through a sterile fitted filter (0.2 μm). However, itis also possible to provide for the plasma to be removed through theseptum without carrying out a centrifugation. The plasma is then, forexample, reinjected into a nerve root or a joint of the patient.

EXAMPLE 2

[0042] Production and Use of a Plastic Syringe With Granules

[0043] In this example, there is use of sterile granules made ofpolystyrene, glass or another modifiable and/or sterilizable material.The surface of the granules is modified using a commercially availablechromic/sulfuric acid product in the batch method as stated inexample 1. The granules are then rinsed with water in order to wash offthe residues of chromic/sulfuric acid. The granules are then incubatedat 121° C. under a pressure of 2 bar for at least 20 min so that thegranules are sterilized and saturated with water. The granules are thendried at 80° C. for 20 min.

[0044] A conventional, unmodified polypropylene syringe new ex-factoryand in the original pack (50 ml, Becton Dickinson, Hetdelberg, Art. No.00137) is charged with the modified and sterilized granules (1, 2, 4 or10 cm³) and with a sufficient amount of an anticoagulant such as heparin(Liquemin, heparin sodium 2500 I.U.) or citrate (for example ACDA).

[0045] The charged syringe, including removal cannula and tubing, ispackaged and then sterilized with gamma rays or electrons.

[0046] The user removes the sterile kit and removes blood from thepatient. The syringe has on its orifice in the closure attachment aseptum which, for the removal, is pierced by the removal accessory, thatis to say the needle of the adapter. After removal of the adapter, theseptum closes automatically again. After removal of blood, the syringeplunger is broken off at a predetermined breaking point.

[0047] The syringe with blood is incubated at 37° C. to 41° C. for 24hours.

[0048] a) If the incubation takes place vertically, the plasma isremoved through the septum and a sterile fitted filter, for example 0.2μm.

[0049] b) If the incubation takes place horizontally, aftercentrifugation of the syringe the plasma is removed through a sterilefitted filter, for example 0.2 μm.

[0050] Reinjection of the plasma takes place, for example, at a nerveroot, into the joint or into the intervertebral disk.

EXAMPLE 3

[0051] Production and Use of a Syringe Without Granules

[0052] A syringe in the original pack, new ex-factory and made of amodifiable, sterilizable material (5, 10, 20 or 50 ml) is modified withchromic/sulfuric acid as indicated in example 1 and then autoclaved anddried. The syringe preferably consists of glass, polystyrene or aspecially modifiable other material.

[0053] The modified and sterilized syringe is charged with a sufficientamount of heparin (Liquemin, heparin sodium 2500 I.U.) or citrate(ACDA).

[0054] The charged syringe, including removal cannula and tubing, ispackaged and then sterilized with gamma rays or electrons.

[0055] The user removes the sterile kit and removes blood from thepatient. The syringe has on its orifice in the closure attachment aseptum which, for the removal, is pierced by the removal accessory, thatis to say the needle of the adapter. After removal of the adapter, theseptum closes automatically again. After removal of blood, the syringeplunger is broken off at a predetermined breaking point.

[0056] The syringe with blood is incubated at 37° C. to 41° C. for 24hours.

[0057] a) If the incubation takes place vertically (for example in atest tube holder), the plasma is removed through the septum, filteringthrough a sterile fitted filter, for example 0.2 μm.

[0058] b) If the incubation takes place horizontally, aftercentrifugation of the syringe the plasma is removed through the septum,filtering through a sterile fitted filter, for example 0.2 μm.

[0059] Reinjection of the plasma takes place, for example, at a nerveroot, into the joint or into the intervertebral disk.

EXAMPLE 4

[0060] Production of the Interleukin 1 Receptor Antagonist in a SyringeUsing Heparin

[0061] A commercially available glass syringe new ex-factory and in theoriginal pack was filled with chromic/sulfuric acid and incubated atroom temperature for 20 min as indicated in example 1. The syringe wasthen rinsed four times with distilled water, packaged, autoclaved at131° C. under a pressure of 2 bar for 30 min and dried at 100° C. for 30min.

[0062] After completion of the modification and sterilization, thesyringe is stored for the time being. Heparin approved under the drugslegislation (Liquemin, heparin sodium 2500 I.U.) is drawn up into thesyringe as anticoagulant.

[0063] The coated syringe is then used to take venous blood from thepatient under sterile conditions.

[0064] The syringe is incubated at room temperature for 12 to 72 hours.During this time the proteins present in the plasma, in particular theinterleukin 1 receptor antagonist, becomes highly enriched in the bloodplasma. It was possible to find a concentration of from 1 to 50 ng/ml ofthe interleukin 1 receptor antagonist.

[0065] The blood or the plasma is then injected into the patient withthe coated syringe.

EXAMPLE 5

[0066] Production of the Interleukin 1 Receptor Antagonist in a SyringeWithout Using Heparin

[0067] A commercially available glass syringe new ex-factory and in theoriginal pack was filled with chromic/sulfuric acid and incubated atroom temperature for 20 min as indicated in example 1. The syringe wasthen rinsed four times with distilled water, packaged, autoclaved at131° C. under a pressure of 2 bar for 30 min and dried at 100° C. for 30min.

[0068] After completion of the modification and sterilization, thesyringe is stored for the time being.

[0069] The coated syringe is then used to take venous blood from thepatient under sterile conditions.

[0070] The syringe is incubated at room temperature for 12 to 72 hours.During this time the proteins present in the plasma, in particular theinterleukin 1 receptor antagonist, becomes highly enriched in the bloodplasma. It was possible to find a concentration of from 1 to 50 ng/ml ofthe interleukin 1 receptor antagonist.

[0071] The diluted blood or the culture supernatant is then injectedinto the patient.

EXAMPLE 6

[0072] Production of IL-1Ra in a Polystyrene Microtiter Plate WithGranules

[0073] The granules are produced by modifying the granules newex-factory and in the original pack in the batch method with the aid ofa commercially available chromic/sulfuric acid product by putting thegranules in a container (such as, for example, a 250 ml glass beakerXXX) to which a 50% strength chromic/sulfuric acid product (Merck,Darmstadt, Order No. 1.02499.2500, chromic/sulfuric acid is diluted withBiochrom ultrapure water No. L 0040 to the desired dilution), and thegranules are treated and thus cleaned and modified. The granules areincubated with the chromic/sulfuric acid for 5 to 60 minutes.

[0074] The chromic/sulfuric acid is then removed and the spheres arewashed thoroughly two to ten times with fresh ultrapure water. Washingis continued until, in the penultimate washing step, the pH and theconductance of the ultrapure water and the washing water are identical.

[0075] The granules are then autoclaved in a glass beaker (such as, forexample, a 250 ml glass beaker XXX) at 132° C. under 2 bar for 30 min(Wolf Autoclave HRM 242 II) and dried at 60° C. to 100° C., preferablyat 80° C., for 30 min. Alternatively, the granules are heat-sterilized(Melag dry sterilizer) directly at 120° C. to 210° C., preferably at180° C., for 30 minutes.

[0076] The following are used for the removal of blood: Sarstedtmonovettes with EDTA, citrate, CPDA, CPDM or heparin, in order toprevent coagulation of the blood taken up later.

[0077] Three to 12 glass beads are placed in each well of a microtiterplate (Nunc, Art. No. 150 687), and 1 ml of blood is added as quickly aspossible after removal.

[0078] After incubation (37° C., 5% CO₂) for 24 h (Hereaus incubator):

[0079] blood clot sediments overnight, and 300 μl of serum are removed,without disturbing or picking up solid constituents, and the IL-1Raprotein concentration is determined (ELISA, R&D, Wiesbaden, QuantikineHuman IL-1Ra).

[0080]FIG. 2: IL-1Ra production in a glass syringe with and withoutglass granules

[0081] Method IL-1Ra production Use of a glass syringe as described inexample 1 Measurements on a patient Glass spheres Roth, treated withchromic/sulfuric acid, washed and autoclaved RB Reaction blank, IL-1Raconcentration on removal (before IL-1Ra production)

[0082] Result

[0083] Addition of the glass granules increases IL-1Ra production

[0084]FIG. 3 IL-1Ra production in a glass syringe with and withoutanticoagulant

[0085] Method IL-1Ra production Use of a glass syringe as described inexample 1 Measurements on a patient Glass spheres Roth, treated withchromic/sulfuric acid, washed and autoclaved CPDM citrate phosphatedextrose mannitol CPDA citrate phosphate dextrose adenine

[0086] Result

[0087] Addition of various anticoagulants influences the efficiency ofIL-1Ra production to varying extents.

[0088]FIG. 4 IL-1Ra production in a microtiter plate with and withoutanticoagulant

[0089] Method IL-1Ra production Use of a microtiter plate as describedin example 6 Measurements on a patient Glass spheres Duran, treated withchromic/sulfuric acid, washed and autoclaved. 12 spheres were added to 1ml of whole blood RB Reaction blank, IL-1Ra concentration on removal(before IL-1Ra production)

[0090] Result

[0091] Addition of various anticoagulants influences the efficiency ofIL-1Ra production to varying extents

[0092]FIG. 5 IL-1Ra production in a glass and plastic syringe with glassgranules in a group of patients

[0093] Method IL-1Ra production Use of a glass syringe as described inexample 1 and 2 Measurements on a group of orthopedic patients Glassspheres Roth, treated with chromic/sulfuric acid, washed and autoclaved

[0094] Result

[0095] IL-1Ra can be produced in both a glass and a plastic syringe, butthe efficiency of IL-1Ra production in a glass syringe is significantlyhigher

[0096]FIG. 6 IL-1Ra production in a glass syringe with and without glassgranules in a group of patients

[0097] Method IL-1Ra production Use of a glass syringe as described inexample 1 Measurements on a group of orthopedic patients Glass spheresRoth, treated with chromic/sulfuric acid, washed and autoclaved

[0098] Result

[0099] IL-1Ra can be produced in a glass syringe both with and withoutglass granules, but the efficiency of IL-1Ra production with glassgranules is significantly higher

[0100]FIG. 7 IL-1Ra production in a glass syringe with and without glassgranules and with and without heparin in a group of patients

[0101] Method IL-1Ra production Use of a glass syringe as described inexample 1 Measurements on a group of orthopedic patients Glass spheresRoth, treated with chromic/sulfuric acid, washed and autoclaved

[0102] Result

[0103] IL-1Ra can be produced in a glass syringe both with and withoutglass granules and with and without heparin, but the efficiency ofIL-1Ra production with glass granules and without heparin issignificantly higher

[0104]FIG. 8 Protein production using a glass syringe with glassgranules in a group of patients

[0105] Method Protein production Use of a glass syringe as described inexample 1 Measurements on a group of orthopedic patients Glass spheresRoth, treated with chromic/sulfuric acid, washed and autoclaved TNFatumor necrosis factor alpha IL-6 interleukin 6 ELISA The IL-1, TNFa andIL-6 concentrations were determined using a Kombo-Kit from R&D

[0106] Result

[0107] Using the described method there is specific production ofIL-1Ra. TNF-alpha and IL-6 production were undetectable. IL-1β is alsoproduced in small quantities. Since the IL-1Ra: IL-1β ratio, which isexpected ought to be more than 100 for a clinical therapeutic effect ofthe produced IL-1Ra (W. P. Arend et al., 1998, Annu. Rev. Immo. 16m27-55) averages 148, the autologously produced IL-1Ra is therapeuticallyvaluable.

[0108]FIG. 9 IL-1Ra production using a microtiter plate with variousamounts of glass granules and with various blood concentrations

[0109] Method IL-1Ra production Use of a microtiter plate as describedin example 6. A difference is, however, that there was not addition of 1ml of heparinized whole blood but addition of sufficient heparinizedwhole blood for the final volume to be 1 ml. Glass spheres Duran,treated with chromic/sulfuric acid, washed and autoclaved 3, 9, 12 =addition of 3, 9 or 12 spheres 1x, 2x, 4x = heparinized whole blooddiluted 1x, 2x or 4x with RPMI 1640 RB Reaction blank, IL-1Raconcentration on removal (before IL-1Ra production) in undiluted blood

[0110] Result

[0111] With every blood dilution an increase in the amount of glassgranules leads to an increase in IL-1Ra production. A maximum for IL-1Raproduction is determined by the ratio between the number of spheres andthe amount of blood, and between the glass surface area and the bloodcell count.

[0112]FIG. 10 IL-1Ra production using a microtiter plate with variousamounts of glass granules and two types of glass granules

[0113] Method IL-1Ra production Use of a microtiter plate as describedin example 6 Glass spheres Duran or Worf, treated with chromic/sulfuricacid, washed and autoclaved 3, 9, 12 = addition of 3, 9 or 12 spheres RBReaction blank, IL-1Ra concentration on removal (before IL-1Raproduction)

[0114] Result

[0115] Increasing the number of spheres or the glass surface area leadsto higher IL-1Ra production both with soda lime glass (Worf as example)and with borosilicate glass (Duran as example)

[0116]FIG. 11 IL-1Ra production using a microtiter plate with variousamounts of glass granules and two types of glass granules

[0117] Method IL-1Ra production Use of a microtiter plate as describedin example 6 Glass spheres Duran, treated with chromic/sulfuric acid,washed and autoclaved RB Reaction blank, IL-1Ra concentration on removal(before IL-1Ra production) Corundum alpha Al₂O₃ suspension Quartz Quartzsand, 220 mg/ml Glass powder 0.2 mg/ml

[0118] Result

[0119] The addition of silicate oxides or aluminum oxides, which areconstituents of the glass granules described (see technical data for theglass granules) , in the form of corundum or quartz sand leads to highIL-1Ra production

[0120]FIG. 12 IL-1Ra production under different container materials

[0121] Method IL-1Ra production Use of a microtiter plate as describedin example 6 Glass spheres Duran, treated with chromic/sulfuric acid,washed and autoclaved RB Reaction blank, IL-1Ra concentration on removal(before IL-1Ra production) PS Polystyrene microtiter plate (from Nunc,Art. No. 150687) PP Polypropylene reaction vessel (from Sarsted, Art.No. 62/554.502) Glass Commercially available reaction vessel, autoclaved

[0122] Result

[0123] IL-1Ra can be produced both in glass and in plastic containers,but the efficiency of IL-1Ra production in glass containers issignificantly higher

[0124]FIG. 13 pH in the washing water after chromic/sulfuric acidtreatment of glass granules

[0125] Method Glass spheres All the listed spheres were treated withchromic/sulfuric acid; the pH was determined using a pH meter after eachwashing step Ultrapure water (Biochrom ultrapure water Art. No. L 0040),pH between 6.0 and 6.5, was used for washing

[0126] Result

[0127] It was possible to wash away all acid residues for all thespheres described after the chromic/sulfuric acid treatment

[0128]FIG. 14 Conductance in the washing water after chromic/sulfuricacid treatment of the glass granules

[0129] Method Glass spheres All the listed spheres were treated withchromic/sulfuric acid, and then the conductance of the washing water wasdetermined after each washing step using a conductance meter Ultrapurewater (Biochrom ultrapure water, Art. No. L 0040), conductance 0 μS, wasused for washing

[0130] Result

[0131] After chromic/sulfuric acid treatment and washing it was notpossible to detect any leachates for any of the spheres described. It isthus possible to preclude IL-1Ra induction by possible leachates withpyrogenic activity. Technical data of the glass granules 1.1 Roth Size2.85-3.3 Material Chem. comp. see below Chem. composition (%) SiO2 68CaO 3 BaO 6 K2O 8 Na2O 10 Al2O3 1 B2O3 2 Lead-free 1.2 SiLi 5506/89-6Size 2.3-2.5 mm Material Borosilicate glass Treatment 1. Grinding method2. Polishing method Chem. composition (%) SiO2 82 Na2O 2 Al2O3 2 B2O3 14Specific gravity (kg/dm3) 223 Mohs hardness 7 Coefficient of linearexpansion 325 (20-300° C.) Hydrolysis class (DIN ISO 719) 1 Acid class(DIN 12116) 1 Alkali class (DIN ISO 695) 2 Transformation temperature (°C.) 530 1.3 SiLi 5004/99-5 Size 2.5 mm Material Soda lime glassTreatment Pressing method Chem. composition (%) SiO2 67 Na2O 16 CaO 7Al2O3 5 B2O3 3 MgO 2 PbO free Mohs hardness >=6 1.4 Worf Size 2 to 3.5mm Material Soda lime glass Treatment Polished and thermally hardenedChem. composition (%) SiO2 65 Na2O 16 CaO 7 Al2O3 5 B2O3 3 MgO 2 Leadcontent zero Density (g/dm3) 2.54 Mohs hardness 6 Hydrolysis class 3Deformation temperature (° C.) 530 +/− 10 1.5 Duran Size 2 to 3.5 mmMaterial Borosilicate glass Treatment 1. grinding method 2. polishingmethod Chem. composition (%) SiO2 81 Na2O + K2O 4 Al2O3 2 B2O3 13Density (g/dm3) 2.23 Coefficient of linear expansion 3.25 (20-300° C.)Hydrolysis class (DIN ISO 719) 1 Acid class (DIN 12116) 1 Alkali class(DIN ISO 695) 2 Transformation temperature (° C.) 525

1. A method for producing IL-1Ra in a syringe, where the syringe isfilled with a body fluid from an organism and incubated, and the IL-1Rais formed in the body fluid.
 2. The method as claimed in claim 1, wherethe syringe consists of glass, plastic, corundum and/or quartz orcontains this or these.
 3. The method as claimed in claim 2, where theplastic is polystyrene, polyvinyl chloride, polyethylene orpolypropylene.
 4. The method as claimed in any of claims 1, 2 or 3,where the syringe has a modified internal structure.
 5. The method asclaimed in any of claims 1 to 4, where the syringe has an internalstructure modified by means of a corrosive agent.
 6. The method asclaimed in any of claims 1 to 3, where the syringe has an unmodifiedinternal structure.
 7. The method as claimed in any of the precedingclaims, where the internal structure of the syringe is formed by itsinternal surface, in particular formations enlarging the surface area.8. The method as claimed in any of the preceding claims, where theinternal structure is formed by beads, spheres, gels, wool, powder,granules or particles made of glass, plastic, corundum and/or quartzpresent in the syringe.
 9. The method as claimed in any of claims 4 to8, where the internal structure consists of polystyrene, polyvinylchloride, polyethylene, polypropylene or contains this.
 10. The methodas claimed in any of the preceding claims, where the corrosive agent isan alkali or acid, in particular chromic/sulfuric acid.
 11. The methodas claimed in any of the preceding claims, where the body fluid isblood.
 12. A method for producing a syringe suitable for in vitroinduction of IL-1Ra, where the syringe is filled with a corrosive agentand incubated, and the agent is removed.
 13. The method as claimed inclaim 12, where the internal structure of the syringe consists ofpolystyrene, polypropylene, polyvinyl chloride, polyethylene, corundum,quartz or glass or contains this.
 14. The method as claimed in either ofclaims 12 or 13, where the corrosive agent is an alkali or acid, inparticular chromic/sulfuric acid.
 15. The method as claimed in any ofclaims 12 to 14, where, after removal of the corrosive agent, theinternal structure of the syringe is washed and, where appropriate,sterilized.
 16. A syringe, in particular produced as claimed in any ofclaims 12 to 15, characterized in that the internal structure of thesyringe consists of plastic, in particular polystyrene, polyvinylchloride, polyethylene or polypropylene, corundum, quartz or glass, orcontains this, and has been treated by means of a corrosive agent togenerate a modified surface of the internal structures.